This invention was made with government support under Grant No. 1R55DK44900-01 awarded by the National Institute of CHD. The government has certain rights in the invention.
This invention relates to a method of controlling growth of microorganisms. More particularly, this invention relates to a method of controlling growth of fungi, such as Candida albicans, and bacteria, such as mycobacteria and xanthomonads, by modulating the activity of an endogenous chorionic gonadotropin-related protein produced by such microorganisms.
Candida is a genus of imperfect fungi characterized by two principal growth habits, yeast cells (or blastospores) and hyphae (or mycelia). It is the only fungus that is commonly part of the normal flora of the skin, mouth, intestinal tract, and vagina, but can cause a variety of infections, including candidiasis, onychomycosis, tinea corporis, tinea pedis, vaginitis, and thrush. C. albicans is the usual pathogen, but other species may also cause infection. Candidiasis is usually a superficial infection of the moist cutaneous areas of the body and most commonly involves the skin, oral mucous membranes, respiratory tract, and vagina. Rarely, there is a systemic infection or endocarditis. Pregnancy predisposes women to infection by C. albicans. E.g., O. S. Kinsman et al., Effect of Mammalian Steroid Hormones and Luteinizing Hormone on the Germination of Candida albicans and Implications for Vaginal Candidosis, 31 Mycoses 617 (1988). Opportunistic infections have become increasingly frequent with the expanding population of immunocompromised individuals.
Hyphal or mycelial forms of Candida albicans are more associated with pathogenicity in humans and animal models than the yeast forms. F. C. Odds, Mozphogenesis in Candida with special reference to Candida albicans, in Candida and Candidosis 22-61 (1988). The mycelial form is invariably seen in smears, scrapings, or histological sections from lesions due to C. albicans, and blastospores produce mycelium in tissues within hours of inoculation into experimental animals. Little is known about the biochemical and molecular basis of the, transition of C. albicans from yeast to hyphae, and genes unique to either of the forms have not yet been isolated. M. B. Kurtz, Dimorphism and high-frequency switching in Candida albicans, in The Genetics of Candida 57-75 (D. R. Kirsch, R. Kelly, M. B. Kurtz eds., 1990). However, environmental changes are well known to influence transition in C. albicans, and several enhancers and inhibitors of transition have already been described. F. C. Odds, Morphogenesis in Candida with special reference to Candida albicans, in Candida and Candidosis 22-61 (1988). Of particular interest is a description of an autoregulatory substance produced by C. albicans, called morphogenic autoregulatory substance, produced by cultures of high concentrations of yeast (10.sup.8 yeast/ml), and reported to inhibit transition in C. albicans. K. C. Hazen & J. E. Cutler, Autoregulation of Germ Tube Formation by Candida albicans, 24 Infect. Immun. 661 (1979). This substance has not been characterized biochemically and only its activity in culture media was described.
A resurgence in the frequency of occurrence of tuberculosis has occurred in recent years, especially among persons with weakened immune systems. Tuberculosis has become one of the major opportunistic infections plaguing patients with acquired immune deficiency syndrome (AIDS). Tuberculosis is caused by species of Mycobacterium, principally M. tuberculosis. Drug-resistant strains have appeared recently, making treatment very difficult.
Human chorionic gonadotropin (hCG) and human luteinizing hormone (hLH) are glycoprotein hormones from a family also containing human follicle stimulating hormone (hFSH) and human thyroid stimulating hormone (hTSH). These hormones are composed of common .alpha.-subunits and unique .beta.-subunits. For example, the .beta.-subunits of hCG and hLH differ by the presence of a 31 amino acid carboxyl-terminal ("carboxyl-tail") region in hCG that is absent in hLH, but have 80% amino acid sequence identity in the remainder of the .beta.-subunit. Recently, the effects of human chorionic gonadotropin (hCG), human luteinizing hormone (hLH), and an hCG-like protein (xCG) isolated from Xanthomonas maltophilia have been described on transition in C. albicans. O. Caticha et al., Stimulation of Candida Albicans Transition by Human Chorionic Gonadotropin and a Bacterial Protein, 18 Endocr. Res. 133 (1992). All three proteins stimulated transition with a minimal dose of 100 ng/ml, and also bound with high affinity nanomolar K.sub.d S to C. albicans blastospores. Specific hCG and hLH binding, as well as the effects of hLH and steroids in the transition of C. albicans, have also been previously published. T. A. Bramley et al., Specific, High Affinity Binding Sites for Human Luteinizing Hormone and Human Chorionic Gonadotrophin in Candida Species, 167 Biochem. Biophys. Res. Commun. 1050 (1990); O. S. Kinsman et al., Effect of Mammalian Steroid Hormones and Luteinizing Hormone on the Germination of Candida Albicans and Implications for Vaginal Candidosis, 31 Mycoses 617 (1988). An immunologic cross-reaction is exhibited between the 48.5 kD xCG protein and the .beta.-subunit of hCG. S. Grover et al., Isolation of a 48.5 kDa Membrane Protein for Pseudomonas maltophilia which Exhibits Immunologic Cross-Reaction to the .beta.-Subunit of Human Chorionic Gonadotropin, 128 Endocrinology 3096 (1991). When added to cultured X. maltophilia, xCG causes a dose-dependent change in both the morphology of the bacteria and the cell density at the stationary stage of the growth cycle. The same bacteria possess a surface binding site that binds hCG, but not hLH, D. T. Carrell & W. D. Odell, A Bacterial Binding Site Which Binds Human Chorionic Gonadotrophin but Not Human Luteinizing Hormone, 18 Endocr. Res. 51 (1992). This site is the only known high affinity binding site in nature that distinguishes hCG from hLH. More recently, an hCG-like protein has been characterized from C. albicans. O. Caticha et al., Characterization of a human chorionic gonadotropin-like protein from Candida albicans, 132 Endocrinology 667 (1993). This 68 kD protein, termed Candida albicans chorionic gonadotropin-like protein (CaCGLP), appears to be a potent inducer of transition. It is presumed to represent an endogenous transition-inducing substance. This protein reacted with the following hCG immunoassays: a polyclonal rabbit anti-hCG equilibrium assay, a carboxy-tail hCG equilibrium assay, two hCG equilibrium assays using monoclonal antibodies, a free .alpha.-subunit equilibrium assay using a monoclonal antibody, and an ultrasensitive immunoradiometric assay for hCG that does not cross-react with hLH or the free .beta.-subunit of hCG. In a specific hLH immunoradiometric assay, however, CaCGLP showed no reaction. When CaCGLP was tested in a transition assay containing 4% rat serum, the protein was 100-fold more potent than hCG in producing transition in C. albicans.
CG-like substances have also been found using immunological methods on the surface of strains of Streptococcus species (ATCC 12818), Staphylococcus species (ATCC 19433, 27848), Enterococcus coli (E. coli, ATCC 25922), and X. maltophilia (ATCC 13637) in efforts to establish a link between human cancers and the production of CG-like substances by bacteria, since these bacteria were obtained from patients with cancer.
In view of the foregoing, it will be appreciated that a method of controlling growth of microorganisms by modulating the activity of a growth-regulating CG-like protein produced by these microorganisms would be a significant advancement in the art.